ZV004 Development of an ELISA for serodiagnosis of Leptospirosis and additional detection of pathogenic Leptospira by PCR

Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Kurzvortr~ge

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T h e o c c u r r e n c e of e n t e r o p a t h o g e n i c bacteria and Cryptosporidium in s e m i d o m e s t i c a t e d R e i n d e e r in F e n n o s c a n d i a - a risk to h u m a n health?

Kemper, N.1; Aschfalk, A } ; H~ller, C }

~Christian-AIbrechts-University; Institute of Hygiene and Environmental Medicine 2The Norwegian School of Veterinary Science; Department of Veterinary Medicine The information about pathogens excreted by semidomesticated reindeer (Rangifer tarandus tarandus) that might represent a health risk to humans is insufficient. Infections may be accomplished through consumption of contaminated reindeer meat or through contamination of the environment due to faecal shedding. The objectives of this study are to figure out the occurrence and prevalence of important enteropathogenic, zoonotic bacteria and parasites in reindeer and to provide recommendations for human health risk calculations. Faecal material from 2243 clinically healthy, semidomesticated reindeer from northern regions of Finland and Norway was sampled. Subsequently, samples were examined for important enteropathogenic bacteria and Cryptosporidium following standard procedures. Enteropathogens were found in 2224 (99.2%) reindeer faecal samples. Escherichia coli were isolated in 2123 (94.7%), Enterococcus spp in 2084 (92.9%), Yersinia spp in 127 (5.7%) and Campylobacter sp, identified as C. hyointestinalis, in one sample only (0.04%). There was no evidence of the occurrence of Salmonella spp, and Cryptosporidium. This study clearly shows that E. coil and Enterococcus spp. belong to the normal intestinal flora of healthy reindeer. However, only certain strains possess the ability to cause severe health problems in humans and also animals, and thus, further analysis of virulence factors was established (e.g. for genes encoding E. coil eae, hly, s t x l , 2 ) ; as done as well to determine pathogenic strains of Yersinia spp. (PCR, biochemical reactions). Even though the public health risk due to reindeer shedding Campylobacter spp,, Salmonella spp. and Cryptosporidium has to be considered very low at present -as proven in this study-, a putative epidemiological threat to human health exists through important potentially zoonotic, enteropathogenic bacteria excreted by reindeer in Fennoscandia. This study was performed as part of the EU-project RENMAN (www. u rova.fi/home/renma n/).

D e v e l o p m e n t of an ELISA for serodiagnosis of Leptospirosis and a d d i t i o n a l d e t e c t i o n of p a t h o g e n i c Leptospira by PCR

Theodoridis, D.1; Gerlach, G}, Homuth, M}; Strutzberg, K. ~

~Tieraerztliche Hochschule; IVD GmbH 2Tier~rztliche Hochschule; Institut f~r Mikrobiologie Introduction: In farm animals Leptospirosis proceeds asymptomatically and causes high economic damage. Since the

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Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Kurzvortr~ge

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identification of the etiologic agent by culture is very difficult, serological diagnosis is of great importance. Serological methods are only able to detect antibodies, directed against the test antigen of the correspondenting serovar. The reference method, the MAT achieves the highest sensitivity (41%). A multi-serovar ELISA with a defined genus-specific antigen, could achieve a higher level of sensitivity and would be time-saving and cost-effective. Identification of Leptospira spp. is possible with different PCR methods, that are based on the amplification of segments of the 16S and 23S RNA sequences. These PCR methods can not assure specifity for pathogenic Leptospira spp. only. A PCR that amplifies a protein-coding sequence occuring only in pathogenic Leptospira spp., could accomplish this demand. Methods and Data: Western-Blot analyses were performed with different preparations of pathogenic and non-pathogenic strains with specific hyperimmune and convalescent sera. An appropriate gene encoding and highly immunogenic protein occuring in all used pathogenic serovars, but not detectable in nonpathogenic, could be recognized by convalescent sera but not by antisera against nonpathogenic Leptospira. With a monospecific antiserum this protein could be identified as LipL41. LipL41 prepared from Leptospira has already been tested successfully in an ELISA. Specific PCR primers for the lip141-gene were selected and PCR was performed with clinical samples and DNA preparations of cultured Leptospira. Conclusions: LipL41 shows to be an interesting candidate for diagnostics of Leptospirosis by ELISA-techniques. A diagnostic PCR could be established, that is able to detect fast and reliably pathogenic Leptospira in different sample material. This project is sponsored by Nieders~ichsisches Ministerium for Wirtschaft, Technologie und Verkehr.

Sensitivity and specificity of different methods (Stamp staining, capture-ELZSA, PCR and cell culture) for the detection of the Q fever agent Coxiella burnetii Henning, K.1; Sting, R?

1Bundesforschungsanstalt f#r Viruskrankheiten der Tiere; Institut fE/r epJdemiologische Diagnostik 2Chemisches und Veterin~runtersuchungsarnt Stuttgart; Serologie Introduction:

Coxiella burnetii is a small, intracellular bacterium which causes abortion in cattle, sheep and goat. These animals are considered to be the reservoir for Q fever infection in humans (zoonosis) which are characterized by flulike illness, hepatitis or endocarditis. But also

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